Extract of toona sinensis from supercritical fluid extraction for treating diabetes and metabolic diseases, the preparation method and the use thereof

ABSTRACT

The  Toona sinensis  extract of the present invention is prepared using supercritical fluid technique, wherein the method includes steps of: (a) drying the leaves of  T. sinensis ; (b) pulverizing the leaves as particles; and (c) extracting the particles with supercritical carbon dioxide to obtain the  T. sinensis  extract. This supercritical  T. sinensis  extract not only can decrease blood sugar level, but also promotes lipid degradation, inhibits the formation of huge lipid droplet and improves the metabolic symptoms. Accordingly, the  T. sinensis  extract further is able to be prepared as food supplement and pharmaceuticals.

FIELD OF THE INVENTION

The present invention relates to a Toona sinensis extract, in particularto an extract of T. sinensis supercritical fluid and the preparationmethod and the use thereof. The supercritical T. sinensis extract canimprove the glucose absorption in vivo, improve the lipid degradationand block the formation of the huge lipid droplet, and thus can bedeveloped as the preventive food, drinks or therapeutic reagents on themetabolic syndromes.

BACKGROUND OF THE INVENTION

Toona sinensis is a Meliaceae defolite arbor, and its leaves can be usedin food and show the medicinal functions on antiphlogosis,detoxification, disinfestations and the treatments on enteritis,dysentery, carbuncle, scabies, tinea capitis, and the improvements onsomatoplasm. In the folk healing in China, the root skin of T. sinensiscan be used to treat leucorrhoea, rheumatism, and gastrointestinaldisorders, and the water with leaves can wash away tinea. T sinensisfurther shows functions on prevention of cold and enteritis.

Chen et al. (2009) indicated that gallic acid extracted from the leavesof T. sinensis has the reactive oxygen species (ROS)-mediated anti-tumoractivity on prostate cancer, and the extraction of T. sinensis leaves ismade by boiling T. sinensis leaves in the water, filtering throughsieves, lyophilizing and extracting with ethanol with variousconcentrations and the lyophilization treatments to obtain differentextracts (Chang et al., 2006; Wang et al., 2008). In addition, U.S.Patent Publication No. 2009/0169658 A1 also indicated that the extractis afforded by the water extraction on T. sinensis leaves and thealcohol processed water extract is further obtained using liquidchromatography with reverse column and is used to inhibit theproliferation of cancer cells. Some food advertisements exaggerate thatthe T. sinensis tea brewed with hot water on the T. sinensis tea bagshas the anti-tumor effect. However, the temperature, vapor and pressureof hot water are not sufficient to leach the components of T. sinensisinto the water, and the dosages of components also do not achieve thetherapeutic dosage and the therapeutic concentration.

Water and various organic solvents have different polarities, and theextraction uses the strength/weakness of the distribution coefficientsof chemicals to the organic solvents to obtain the target extract.Furthermore, partial extracts might be destroyed or lost by thewater-boiling extraction or the organic-solvent extraction, which thusresults in the decrease of yield and recovery. The remaining organicsolvent in the extracts also might harm the users. Therefore, findingthe extraction method with advantages of non-toxicity and non-residueand without decrease of yield/recovery becomes the target in theapplication of T. sinensis.

Obesity, diabetes mellitus and their derived metabolic diseases are theinteresting issues along with the human culture progress, and obesity isthe main reason for the diseases such as cardiovascular disease, insulinresistance, diabetes mellitus and cancer, etc. The currently effectivemanner to decrease blood glucose is the subcutaneous injection ofinsulin. However, it needs to tightly monitor blood glucose and adjustinsulin dosage, and thus it makes the diabetes patients' livesinconvenient. Type II diabetes mainly is treated with oral hypoglycemicagents, which mainly includes four groups, insulin-secreting agents(sulfonylureas and glinides), Metformin, insulin sensitizer (such asthiazolidinedion (TZD)) and α-glucosidase inhibitor. The functionalmechanism and side effects in each group are not the same, and patientsmust try several drugs to find out the sufficient and effective drugs.

Since some oral hypoglycemic agents would induce gastrointestinal sideeffects such as diarrhea to result in patients' repulsion, they cannotbe taken by the patients with liver or kidney disfunction. Further,rosiglitazone of the insulin sensitizer-typed drugs has hazard onincreasing myocardial infarction and fracture in women. Therefore,hypoglycemic agents with more efficiency, less side effects, lower priceand beneficial on liver- or kidney-disfunctioned patients still havenecessity in the international medicinal market.

Although there are various natural plant extracts in the marketannounced to loss weight or reduce blood glucose, the scientificevidences are absent and these extracts cannot be acceptable by theclinical doctors and the international market. Accordingly, the purposesof the present invention is to overcome the drawbacks in theconventional extraction method, efficiently prepare the extracts onanti-diabetes and anti-metabolic diseases.

It is therefore attempted by the applicant to deal with the abovesituation encountered in the prior art.

SUMMARY OF THE INVENTION

For overcoming the drawbacks in the conventional extraction method andpreparing the extracts on treatments of diabetes and metabolic diseases,the present invention uses the supercritical fluid technique to extractthe leaves of T. sinensis to obtain the supercritical T. sinensisextract. The supercritical extract of T. sinensis can efficientlyimprove glucose absorption in vivo, improve lipid degradation, inhibitthe formation of huge lipid droplet and improve metabolic syndromes, andthus can be prepared as food supplement and pharmaceuticals.

Supercritical fluid extraction (SFE) is a method commonly used for theseparation of active compounds from herbs and natural products(Michielin et al., 2009). Supercritical carbon dioxide fluid (SCF), byits non-polarity, is effective in obtaining non-polar components(Yamaguchi et al., 1986). Supercritical carbon dioxide (sc-CO₂) is themost recognized supercritical solvent and is classified as GRAS(Generally Recognized as Safe) by the Food and Drug Administration ofthe United States. Carbon dioxide achieves the critical point of gasphase and liquid phase at 31.7° C. and 72.8 atm. However, at theconditions behind the critical temperature, critical pressure andcritical density, CO₂ represents high solubility and high permeationowned by the supercritical fluids (Wang et al., 2009). Since materialscan be extracted by CO₂ at normal temperature and CO₂ does notchemically react with the extracted materials, the materials stillmaintain their activity after extraction. Furthermore, CO₂ evaporates atnormal temperature and pressure (NTP) after extraction, and thus thefast concentration effect of material can be achieved and the problem ofthe remaining solvent is absent. Thus, supercritical fluid has theeffects on distillation and organic solvent extraction, and theseparation effect of the raw material is achieved by the high diffusioncoefficient, low viscosity, low surface tension of the supercriticalfluid and the density adjustment. The use of sc-CO₂ as “green” solventsfor chemical reactions and separations is currently an active researcharea (Wang et al., 2009). Sc-CO₂, characterized by its non-polarity, iseffective in obtaining non-polar components (Yamaguchi et al., 1986) andis an attractive alternative of organic solvents due to itsnonexplosivity, nontoxicity, chemical inertness, and low cost (Wang &Weller et al., 2008). In addition to the supercritical CO₂ fluid, othersupercritical fluids can be applied in the present invention.

The first aspect of the present invention is to provide a preparationmethod of a T. sinensis extract for reducing a blood glucose level,including steps of: drying leaves of T. sinensis; pulverizing the leafas particles; and extracting the particles with a supercritical carbondioxide to obtain the T. sinensis extract. The leaf includes youngleaves, matured leaves and/or young shoots.

The second aspect of the present invention is to provide a T. sinensisextract for reducing blood glucose level including at least one of amonoterpene, a monoterpene derivative, a diterpene derivative, atriterpene, a sesquiterpene, sequiterpene derivative, a saturated fattyacid, a unsaturated fatty acid, an alkane, an alkene and a phytosterol.

The monoterpene includes α-pinene and β-pinene, the monoterpenederivative includes limonene, the diterpene derivative includes phytol,and the triterpene includes squalene. The sesquiterpene includesα-cubebene, copaene, aromadendrene, caryophyllene, α-caryophyllene,β-caryophyllene, aristolene, γ-patchoulene, α-selinene, β-selinene,γ-selinene, elixnen, germacrene A, germacrene B, germacrene D,β-elemene, γ-elemene, δ-elemene, τ-elemene, γ-cadiene, τ-muurolene,α-farnesene and lepidozene, and the sesquiterpene derivative includescaryophylene oxide, spathulenol, ledene oxide, germacrene D-4-ol andglobulol. The saturated fatty acid includes ethyl pentadecanoate andethyl palmitate, and the unsaturated fatty acid includes methyllinoleate, ethyl 9,12-octadecadienoate and ethyl α-linolenate.

Alkane has a carbon number not less than 21, in particular, alkaneincludes n-heneicosane, n-docosane, n-tricosane, n-pentacosane,n-heptacosane, n-octacosane, n-nonacosane, n-hentriacontane,n-dotriacontane, n-pentatriacontane, n-hexatricontane, n-tritetracontaneand n-tetratetracontane. The alkene includes (17E)-17-pentatriacontene,and the phytosterol includes β-sitosterol. The T. sinensis extractfurther includes 6-methyl-5-hepten-2-one and phytone.

The T. sinensis extract is manufactured as food and/or drugs, andfurther is used for improving metabolic diseases and inhibiting anaccumulation of fats in the body.

The third aspect of the present invention is to provide a preparationmethod of a T. sinensis extract for reducing blood glucose level,including a step of extracting T. sinensis with a supercritical fluid toobtain the T. sinensis extract.

The supercritical fluid includes CO₂ supercritical fluid, and the T.sinensis extract further contains a T. sinensis essential oil. The leafparticles are obtained by sequentially drying and pulverizing the leaf.

The fourth aspect of the present invention is to provide a method fordetecting at least one component in a T. sinensis extract, including astep of determining an amount of the at least one component using GC-MS,and the at least one component contains several groups of chemicals, aswell as 6-methyl-5-hepten-2-one and phytone in the above-mentionedsecond aspect of the invention.

Since there does not have any reference to report the low-polaritymaterial of T. sinensis to own the therapeutic effect of reducing bloodglucose and inhibiting fat accumulation, the supercritical fluid extractof T. sinensis made by CO₂ supercritical fluid in the present inventionis benefit for improving metabolic syndromes, and the extract canovercome the drawbacks in the prior art and thus has industrialusefulness.

The above objectives and advantages of the present invention will becomemore readily apparent to those ordinarily skilled in the art afterreviewing the following detailed descriptions and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the schema showing the CO₂ supercritical fluid extractorfor the T. sinensis supercritical extract in the present invention.

FIGS. 2A to 2B depicts the diagram of the non-polar constituents of T.sinensis extract analyzed by GC/MS.

FIG. 3A shows the remaining glucose concentrations of the media consumedby the differentiated 3T3-L1 cells by the treatment without (control) orwith insulin, rosiglitazone or TS-SCF.

FIG. 3B shows the percentages of glucose consumed by 3T3-L1 adipocyteswith a started concentration of 300 mg/dL.

FIGS. 3C and 3D show the comparison of (C) the medium glucose and (D)the percentage of glucose consumption among the batches of TS-SCFs. InFIGS. 3A to 3D, the data are presented as mean±SD from six independentexperiments, and each sample was performed in duplicate for glucoseconcentration measurement. a, p<0.05 compared to control; b, p<0.01compared to control; c, p<0.001 compared to control.

FIG. 4 depicts the quantification of lipid accumulation in 3TE-adipocytein different treatments. The data are presented as mean±SD from sixindependent experiments, and each sample was performed in duplicate forglucose concentration measurement. a, p<0.05 compared to control; N.S.,no significance compared to control.

FIGS. 5A and 5B are the drawings showing the effects of insulin,rosiglitazone (Rosig), and TSL extracts on (A) the medium glucose and(B) the glucose consumption in 3T3-L1 adipocyte model. In FIGS. 5A and5B, the data are presented as mean±SD from six independent experiments,and each sample was performed in duplicate for glucose concentrationmeasurement. a, p<0.05 compared to control; b, p<0.01 compared tocontrol; c, p<0.001 compared to control.

FIGS. 6A and 6B shows (A) the blood glucose change and (B) the bodyweight change in mice. The feeding of a high-fat diet was started at day−14, and day 0 represented the day of the STZ injection (45 mg/kg). DMrepresented the group of high-fat-diet-fed mice with the STZ injection;DM+TS-SCF represented the group of high-fat-diet-fed mice with the STZinjection plus TS-SCF (150 mg/kg/day) treatment; DM+Rosig representedthe group of high-fat-diet-fed mice with the STZ injection plusrosiglitazone maleate (150 μg/kg/day). Data are mean±SD. a, p<0.001compared to the control, DM+TS-SCF, or DM+Rosig groups.

FIGS. 7A and 7B are the drawings showing the plasma levels of (A)adiponectin and (B) TNF-α both detected by ELISA kits. Data are mean±SD.a, p<0.05; b, p<0.01; N.S., no significance.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more specifically withreference to the following Embodiments. It is to be noted that thefollowing descriptions of preferred Embodiments of this invention arepresented herein for purpose of illustration and description only; it isnot intended to be exhaustive or to be limited to the precise formdisclosed.

Example 1 Supercritical Fluid Extraction for the Leaves of T. Sinensis(TSL)

In order to perform a more extensive phytochemical screening and searchfor novel active compounds of leaves of Toona sinensis, supercriticalfluid extraction (SFE) was used to obtain the non-polar constituents ofTSL.

Method:

The raw material, “the leaves of T. sinensis (TSLs)”, was pretreated forextraction with a cold-air-dried method and ground into 1˜5-mm particleswith a moisture content lower than 30%. To prepare the non-polar TSLextract, a pre-engineered supercritical fluid extraction pilot plant(Fa. NATEX Process Technology GmbH, Ternitz, Austria; extractionpressure up to 1000 bar and utilizable extraction volume at 5 L) wasconstructed. The extraction method and equipment were modified from thereport of Rońyai et al., 1998. The scheme of the extraction procedure isshown in FIG. 1. A total of 1600 g of TSL was weighed accurately andsupplied into the extraction vessel 4 for the extraction of solids. Theextraction vessel was pressurized. Then, liquid CO₂ was flowed into thecooler 2 for precooling and was adjusted prior to the commencement ofthe extraction. Pure CO₂ was compressed by a high-pressure diaphragmpump 3 to 350-550 bar at 40-60° C. and allowed to flow through theextraction vessel at a flow rate of 30-35 kg CO₂ per hour.Supercritical-CO₂ is a solvent that is mainly suitable for transportingnon-polar or weak-polar substances (TS-SCF) into the separator 5, whereit is expanded by valves. After separation, the regenerated CO₂ flowsback into the reservoir 1 and can be used for extraction again. Theextraction time was in the range of 60 to 120 min, and the extractionwas stopped depending on whether the yield was less than 0.1% of the TSLas 30-70 kg CO₂ passed through the vessel 4. To confirm the efficacy ofsupercritical-flow extracted leaves of T. sinensis from differentresources (geographical or seasonal variation), three different batchesof TSLs were collected and extracted using the method mentioned above.After the manufacturing processes, three extracts were harvested andnamed TS-SCF-I, TS-SCF-II and TS-SCF-III, respectively.

Result:

Each batches of supercritical-CO₂ fluid extract of TSL was obtained fromthe separator 5 and processed an water-drying procedure, and therecovery was 14%˜18%. The raw material of EXAMPLE 1 included the youngshoots, young leaves and matured leaves of T. sinensis.

Example 2 GC/MS Analyses of TS-SCF

Method:

Chemical constituents of TS-SCF were analyzed by gas chromatography-massspectrometry (DSQ II Single Quadrupole GC/MS, Thermofisher Scientific,USA) on a 30 m×0.25 mm×0.25 μm DB-5MS capillary column (Agilent J&WScientific). The column oven temperature was programmed as follows: 100°C. (5 min) to 119° C. at 40° C./min, maintained at 119° C. for 26 min,then to 157° C. at 8° C./min, maintained at 157° C. for 5 min, then to181° C. at 40° C./min, immediately heated to 188° C. at 3° C./min, heldfor 23 min, then increased to 230° C. at 5° C./min, held for 10 min,then raised to 242° C. at 5° C./min, held for 5 min, then heated to 280°C. at 1° C./min, and held for 10 min. The other parameters were asfollows: injection temperature, 250° C.; ion source temperature, 250°C.; EI (electron bomb ionization), 70 eV; carrier gas, He at 1 mL/min;injection volume, 5 μL; split ratio, 1:50; and mass range, m/z 45-800.Identification of the compounds was based on a comparison of retentionindices (determined relative to the retention times of n-alkanes on theDB-5MS column) and mass spectra with those of authentic samples, datafrom Wiley/NBS Registry of Mass Spectral Data (V. 5.0)/NationalInstitute of Standards and Technology (NIST) MS Search V. 2.0 and theliterature. The relative percentage of each compound in TS-SCF wasquantified based on the peak area integrated by the analysis program.

Result:

The yield of supercritical-CO₂ extraction from TSLs was 14-18% (w/w)according to the 30% dry weight of raw material. Taking TS-SCF forexample, a total of 24 main constituents of TS-SCF were identified asshown in Table 1, and the chemical profile was presented in FIGS. 2A and2B. Most of the constituents in the TS-SCF extract can be classifiedinto different chemotypes, including monoterpenes and its derivatives,sesquiterpenes and its derivatives, saturated and unsaturated fattyacids, long-chain n-alkenes, phytosterols and others. The components ofanother three batches was listed in Table 1. Since different batches ofTS-SCF were made from T. sinensis planted in different areas in Taiwan,the above-mentioned compounds would not appear in each batch of TS-SCF.

TABLE 1 Total vision of components from every batch of TSLs ComponentsComponents Components Components (TS-SCF-I) Ki^(a) (TS-SCF-II) Ki^(a)(TS-SCF-III) Ki^(a) (TS-SCF) Ki^(a) Monoterpenes MonoterpenesSesquiterpenes Sesquiterpenes α-Pinene 938 α-Pinene 936 Elixene 1199δ-Elemene 1340 β-Pinene 982 δ-Elemene 1341 β-Elemene 1386 SesquiterpenesCopaene 1398 β-Caryophyllene 1417 Monoterpene derivatives β-Elemene 1386β-caryophyllene 1416 Germacrene D 1481 Limonene 1026 Copaene 1389γ-Patchoulene 1441 α-Selinene 1489 γ-Elemene 1437 α-Caryophyllene 1453γ-Selinene 1497 Sesquiterpenes τ-Muurolene 1468 Germacrene D 1483Germacrene A 1508 α-Cubebene 1355 β-Selinene 1483 β-Selinene 1485α-Farnesene 1512 β-Elemene 1389 α-Selinene 1489 γ-Selinene 1497Lepidozene 1528 Copaene 1396 Aromadendrene 1753 Germacrene A 1510Germacrene B 1563 Caryophyllene 1467 Germacrene B 1563 Germacrene D 1481Sesquiterpene derivatives Aristolene 1756 Sesquiterpene derivativesβ-Selinene 1486 Caryophylene oxide 1567 Aromadendrene 1758 GermacreneD-4-ol 1574 τ-Elemene 1488 Spathulenol 1574 Spathulenol 1576 GermacreneA 1506 Ledene oxide 1892 long-chain n-alkanes Globulol 1578 Germacrene B1561 n-Heneicosane 2102 γ-Cadiene 1752 Diterpene derivatives n-Docosane2201 Diterpene derivatives Phytol 1949 n-Tricosane 2308 Phytol 1949Saturated fatty acids 1997 n-Octacosane 2804 Ethyl pentadecanoateSaturated fatty acids n-Hentriacontane 3000 Saturated fatty acids Ethylpentadecanoate 1993 n-Dotriacontane 3201 Ethyl palmitate 1975Unsaturated fatty acids 2091 n-Pentatriacontane 3503 Methyl linoleateUnsaturated fatty acids n-Hexatricontane 3601 Unsaturated fatty acidsMethyl linolenate 2092 n-Tritetracontane 4305 Ethyl9,12-octadecadienoate 2173 n-Tetratetracontane 4400 Ethyl α-linolenate2176 Long-chain n-alkanes n-Heptacosane 2703 Long-chain n-alkanesn-Octacosane 2804 n-Pentacosane 2500 n-Pentatriacontane 3500n-Heptacosane 2700 n-tetratetracontane 4402 n-Nonacosane 2900n-Hentriacontane 3100 Alkenes n-Pentatriacontane 3500(17E)-17-Pentatriacontene 3499 Triterpene Others Squalene 27906-methyl-5-hepten-2-one 984 Phytone 1850 Phytosterols β-Sitosterol 3410^(a)Kovats index relative to n-alkanes (C10-C40) on DB-5MS column andthe identification was based on comparison of the mass spectrum fromdatabase, Kovats index on a DB-5MS column in reference.

Example 3 In Vitro Model for Determination of Anti-Diabetic Effect

Adipose tissue is one of the major sites of postprandial glucose uptake(Watson et al., 2007). To speed up the screening of novel anti-diabeticcandidate materials from natural products, an in vitro model wasestablished by comparing the 24-hour glucose consumption from theculture medium of 3T3-L1 adipocytes.

Method:

The same amount (5×10⁵ cells) of 3T3-L1 preadipocytes (BCRC #60159;Bioresource Collection and Research Center, Taiwan) was seeded andcultured in normal glucose (100 mg/dL) DMEM supplemented with 10% FBS,100 U/mL of penicillin, and 100 μg/mL of streptomycin in a humidifiedatmosphere of 95% air and 5% CO₂ at 37° C. When the cell density reached100% confluence, 3T3-L1 preadipocytes were induced to differentiate bytreating the culture with 450 mg/dL D-glucose, 0.32 μM insulin, 0.5 mM3-isobutyl-1-methylxanthine and 1 μM dexamethasone for two days. Then,the culture medium of the differentiated adipocytes was changed to DMEMcontaining 300 mg/dL D-glucose with or without the administration of TSLextracts. After 24 hours, the anti-diabetic activity was determined bymeasuring the medium glucose concentration using a Roche Cobas Integra400 Chemistry Analyzer (Roche Diagnostics, Taipei, Taiwan). Thecoefficient of variation (CV) of the analyzer was 0.62-0.92% within-runand 1.1-1.2% between days. To confirm whether the in vitro model wassufficient to measure the glucose-lowering effect, insulin androsiglitazone were used as positive controls.

Result:

As shown in FIG. 3A, the medium glucose concentration was decreased byapproximately 18 mg/dL in the control group without the addition of anyanti-diabetic reagent after 24 hours, with a starting glucoseconcentration of 300 mg/dL. When converted to the percentage of glucoseconsumption, only about 6% of the glucose in the medium was used by3T3-L1 adipocytes (FIG. 3B). Insulin (i.e., 3.2×10⁻⁷ to 3.2×10⁻⁵ M)decreased by approximately 46-57 mg/dL, and rosiglitazone (abbreviatedas “Rosig”, i.e., 12.5 to 50 μg/mL) decreased by approximately 50-80mg/dL of the glucose concentration in the media (FIG. 3A). By convertingthe data to percentage of glucose consumption, insulin and rosiglitazoneincreased the use of medium glucose to 15-18% and 16-27%, respectively(FIG. 3B). Compared to the control group, insulin and rosiglitazonesignificantly decreased the glucose concentrations of the media in adose-dependent manner in the 3T3-L1 adipocyte model, indicating thatthis in vitro model could be used as a quick screening method for novelanti-diabetic reagents that target adipocytes. Through this in vitromodel, it was observed that TS-SCF possessed an excellent anti-diabeticeffect. The glucose-lowering effect of 12.5 μg/mL of TS-SCF was equal tothat of 50 μg/mL of rosiglitazone (FIG. 3A). A total of 50 μg/mL ofTS-SCF could increase the use of medium glucose to approximate 50% (FIG.3B). As shown in FIGS. 3C and 3D, the administration of every batch ofTS-SCF (50 μg/mL) could significantly decreased the glucoseconcentrations of the media and possessed an excellent anti-diabeticeffect in our 3T3-L1 adipocyte model.

Example 4 TS-SCF Blocked Adipogenesis in Adipocytes

Method:

Lipid droplets were detected by Oil Red O staining (Kinkel et al.,2004). 3T3-L1 cells were washed three times with PBS and fixed in 10%formalin for 1 hour. After washing with PBS, the cells were stained withOil Red O staining solution (0.5% Oil Red O in 100% propylene glycol).After 1 hour, cells were washed three times with water and observedunder a phase contrast microscope. To quantify the cellular lipidquantity, stained cells were incubated with 100% isopropanol and shakenat room temperature for 20 minutes, and then Oil Red O in thesupernatant was measured at 490 nm by an ELISA reader.

Result:

On day 6, a significant increase in lipid droplet formation was observedby Oil Red O staining in the differentiated cells relative to theundifferentiated cells. The formation of lipid droplets was almostcompletely blocked when TS-SCF was added to the induction medium, theDMSO treated group was blank (data not shown). After extracting the OilRed O from the cells, a serious increase in lipid accumulation wasobserved in the differentiated group (FIG. 4). In contrast, inducedcells that were co-treated with 50, 100 μg/mL TS-SCF showed significantincreases in lipid formation, as compared to the undifferentiated group(FIG. 4). These results indicate that inhibition of TS-SCF obstructedadipogenesis in adipocytes.

Example 5 Anti-Diabetic Effect of Extracts of High to Mid/High PolarComponents of TSL Differed from TSL-SCF

As the described above, supercritical fluid extract of TSL (TS-SCF)exhibited hypoglycemia effect and inhibitory action of lipidaccumulation during differentiation of 3T3-L1 preadipocytes, and thecomponents was identified as non-polar compositions in the presentinvention. To compare with the prior art that mentioned the effectivechemical components of TSL extracts have so far shown a distribution inthe range from high to mid-high polarity (e.g., aqueous extract, aqueoussolution processed by using 99.5% to 25% of ethanol, methanol extract,and aqueous extract of fermented Toona sinensis), three differentextraction methods were generalized to confirm the anti-diabetic effectof the extracts, respectively.

Method:

The methods of in vitro model for determination of anti-diabetic effectare the same as the contents described in EXAMPLE 3. To obtain the highto mid-high polar components of TSL, three different extraction methodswere used to produce the following extracts, which were previouslyreported (Liao et al., 2007; Hsieh et al., 2005; Yang et al., 2003).TSLs were (i) extracted by reverse-osmosis of water (1:4 w/v) by boilingfor 30 min and then cooling down without further boiling for two hoursat room temperature (Liao et al., 2007); (ii) extracted by soaking in100° C. boiled water (1:10 w/v) three times, each time for 30 min (Hsiehet al., 2005); and (iii) extracted by boiling in 50% v/v alcohol/waterfor three hours (Yang et al., 2003). Next, the leaves were removed andthe remaining liquids were filtered through filter paper (Advantec,Tokyo, Japan). Afterward, the filtrated solutions were concentratedusing an evaporator (rotary vacuum evaporator, vv2000; Heidolph,Schwabach, Germany) under reduced pressure and three crude extracts wereobtained, which were named TS-WB (i), TS-WS (ii), and TS-E (iii); theyield of each extract was 6.03%, 5.13%, and 8.36%, respectively.

Result:

FIGS. 5A and 5B show the comparison of glucose-lowering effects amongthe non-polar TSL extract (i.e., TS-SCF) and the high to mid-high polarTSL extracts (i.e., TS-E, TS-WS, and TS-WB). The results demonstratethat only the TS-SCF significantly reduced the glucose in the mediaafter the adipocytes were treated with an equal concentration (i.e., 50μg/mL) of each TSL extract for 24 hours (FIG. 5A). Equal volume ofsolvents (100% EtOH or 100% DMSO) that were used to dissolve thereagents showed no influence on medium glucose consumption in 3T3-L1adipocytes. FIG. 5B shows the data converted to the percentage ofglucose consumption.

Example 6 Determination of Anti-Diabetic Effect In Vivo

Method:

Eight-week-old male C57BL/6J mice were obtained from BioLASCO Technology(Charles River Taiwan Ltd.). All of the mice received standard animalcare under supervision of the Institutional Animal Care and UseCommittee of Kaohsiung Medical University, Taiwan. The mice were cagedin an air-conditioned animal facility at 23° C. on a 12-h light/darkcycle and were maintained with free access to water and food. Animalswere fed either a normal chow diet consisting (as a percentage of totalkcal) of 11% fat, 65% carbohydrate, and 24% protein (Maintenance diet1320, Altromin Spezialfutter GmbH & Co. KG, Germany) or a high-fat dietconsisting of 45% fat, 35% carbohydrate, and 20% protein (D12451,Research Diets, Inc., New Brunswick, N.J., USA). After two weeks oneither diet, mice were divided into four groups: (i) control (n=7); (ii)high-fat-diet mice with STZ injection (DM; n=5); (iii) high-fat-dietmice with STZ injection plus TS-SCF treatment (DM+TS-SCF; n=6); and (iv)high-fat-diet mice with STZ injection plus rosiglitazone treatment(DM+Rosig; n=4). The mice in groups ii-iv were injected with STZ (45mg/kg), and both STZ-injected and non-injected animals were kept ontheir original diets (chow or high-fat) for the duration of the study.TS-SCF (150 mg/kg/day) and rosiglitazone (150 μg/kg/day) treatments viagastric gavage were begun two days after the STZ injection. Therosiglitazone maleate used in this experiment did not contain anyinactive ingredient and the pure compound was kindly provided byGlaxoSmithKline, Ltd. (Taiwan).

Throughout the experiment, body weight and blood glucose from the tailtip were monitored weekly. Blood glucose was detected by the ACCU-CHEKblood glucose meter (Roche Diagnostics, Taipei, Taiwan). Animals weresacrificed after eight weeks of STZ injection and were euthanized byintraperitoneal injection with the anesthetic Zoletil (10 mg/kg)(Virbac, Carros, France). Blood samples were collected from the heart atthe time of sacrifice for the measurement of biochemistry data using aRoche Cobas Integra 400 Chemistry Analyzer (Roche Diagnostics, Taipei,Taiwan).

Insulin from plasma were detected by ELISA kits (Crystal Chem, Inc.Downers Grove, USA). The homeostasis model assessment of insulinresistance (HOMA-IR) was calculated using the following formula: fastingblood glucose (mg/dL)×fasting insulin (μU/mL)/405.

Result:

After the evaluation of glucose-lowering potential via our in vitromodel, the anti-diabetic effect of TS-SCF was further confirmed by thetype 2 diabetic mouse model. FIG. 6A shows that the blood glucosesignificantly increased 14 days after the low-dose STZ injection in theDM group compared to that of the control group, the DM withTS-SCF-treated group, or the DM with rosiglitazone-treated group. Theblood glucose level continuously increased to 584.60±87.65 mg/dL 56 daysafter the injection with STZ. In contrast, TS-SCF and rosiglitazonesignificantly prevented the progression of diabetes and controlled theglucose level between 200 and 300 mg/dL during the experimental period(FIG. 6A). FIG. 6B shows that there was no significant difference in thebody weight change among these four groups.

Table 2 summarizes the biochemical data of the mice. There was nosignificant difference in the gains in body weight among the four groups(p=0.7122). Relative to the control group, the fasting plasma glucosesignificantly increased in the DM group (p<0.001). In comparison withthe DM group, the TS-SCF and the rosiglitazone groups showedsignificantly lower fasting glucose levels (Table 2). The fasting plasmainsulin levels showed no significant difference among the groups(p=0.1688). However, the HOMA-IR (homeostasis model assessment forinsulin resistance) was significantly higher in the DM group (p<0.001compared to the control group). The treatments of TS-SCF androsiglitazone showed to have notably lower HOMA-IR values (p<0.001compared to the DM group), indicating that TS-SCF and rosiglitazonecould maintain insulin sensitivity in the mice. In addition topreventing the progression of diabetes, TS-SCF, as with rosiglitazone,significantly decreased the plasma triglycerol level, which was elevatedin the DM group (Table 2). However, TS-SCF and rosiglitazone reduce thetotal plasma cholesterol level was not observed (Table 2). The plasmaALT level was increased in the DM group; in contrast, TS-SCFsignificantly prevented the elevation of plasma ALT levels (Table 2).Compared to the control group, the plasma levels of ALT and creatininewere not elevated by the TS-SCF treatment, indicating that TS-SCF had nohepatic or renal toxicity throughout the eight-week administration.

TABLE 2 Biochemical data of mice. Control DM DM + TS-SCF DM +Rosiglitazone p-value Body weight gain (g)  6.70 ± 1.50  7.47 ± 1.77 7.52 ± 1.98  8.10 ± 1.95 0.7122 Fasting glucose (mg/dL) 155.14 ± 21.30484.60 ± 87.65^(a) 290.00 ± 65.55^(b) 205.00 ± 84.87^(b) 0.0001 Insulin(ng/mL)  1.45 ± 0.76  1.31 ± 0.71  0.66 ± 0.51  0.98 ± 0.77 0.1688HOMA-IR  15.92 ± 1.15  44.83 ± 4.39^(a)  13.47 ± 2.38^(b)  14.29 ±4.60^(b) 0.0001 Triglycerol (mg/dL)  72.87 ± 13.70  94.42 ± 16.70^(c) 63.27 ± 5.34^(d)  64.94 ± 4.82^(d) 0.0017 T-CHO (mg/dL)  87.32 ± 6.57130.01 ± 10.60^(a) 138.68 ± 16.00 137.65 ± 32.62 0.0001 HDL-C (mg/dL) 76.23 ± 6.87 110.72 ± 7.91^(a) 125.95 ± 9.19^(e) 126.03 ± 32.17 0.0001LDL-C (mg/dL)  5.25 ± 3.21  10.21 ± 0.96^(f)  8.03 ± 1.14  9.81 ± 8.290.1576 ALT (U/L)  39.76 ± 7.17  50.62 ± 8.13^(f)  38.30 ± 6.86^(e) 62.00 ± 27.00 0.0058 Creatinine (mg/dL)  0.48 ± 0.47  1.53 ± 0.64^(c) 0.71 ± 0.40^(e)  0.54 ± 0.18^(e) 0.0081 Values are mean ± SD, and datawere analyzed by one-way analysis of variance and the Bonferroni testHOMA-IR: homeostasis model assessment for insulin resistance, T-CHO:total cholesterol, HDL-C: HDL cholesterol, LDL-C: LDL cholesterol, andALT: alanine aminotransferase ^(a)p < 0.001 compared to control ^(b)p <0.001 compared to DM ^(c)p < 0.05 compared to control ^(d)p < 0.01compared to DM ^(e)p < 0.05 compared to DM ^(f)p < 0.01 compared tocontrol

Example 7 Morphological Evaluation of Adipose and Hepatic Tissues

Method:

A sample of adipose and hepatic tissues from each mouse was fixed in 4%formaldehyde and embedded in paraffin wax. Sections were stained withhematoxylin and eosin (H&E) and observed using a microscope. Themorphological evaluation of was performed by hematoxylin and eosinstains.

Result:

The cell size of adipocytes in the DM group was enlarged compared tothat of the control group. Relative to the DM group, administration ofTS-SCF could reduce the cell size. In contrast, the rosiglitazonetreatment maintained cell sizes as large as those of the DM group. Thehepatosteatosis in the DM mice was characterized by balloonedhepatocytes. The administration of TS-SCF and rosiglitazone couldeffectively prevent the hepatosteatosis.

Example 8 Effect of Ts-SCF on Adiponectin and TNF-α Production in Type 2Diabetic Mouse Model

Adiponectin is a protective adipokine that has anti-inflammatory andanti-atherosclerotic effects; a deficiency of adiponectin has beenassociated with obesity-related disorders such as diabetes andcardiovascular disease (Kiess et al., 2008). TNF-α, a proinflammatorycytokine, is expressed in and secreted by adipose tissue, its levelscorrelating with the degree of adiposity and associated insulinresistance. Targeting TNF-α and/or its receptors has been suggested as apromising treatment for insulin resistance and type 2 diabetes (Chen etal., 2010).

Method:

Adiponectin, and TNF-α from plasma were detected by ELISA kits(MyBioSource, LLC, San Diego, USA).

Result:

As shown in FIG. 7A, plasma adiponectin was decreased in the DM grouprelative to the control group (p<0.05). Treatment with TS-SCF couldsignificantly reverse the decrease of plasma adiponectin (p<0.01compared to the DM group). The plasma TNF-α level was not significantlyincreased in the DM group after our 8-week experimental period, and thevalues were not significantly different among the four groups (FIG. 7B).

While the invention has been described in terms of what is presentlyconsidered to be the most practical and preferred Embodiments, it is tobe understood that the invention needs not be limited to the disclosedEmbodiments. On the contrary, it is intended to cover variousmodifications and similar arrangements included within the spirit andscope of the appended claims, which are to be accorded with the broadestinterpretation so as to encompass all such modifications and similarstructures.

1. A preparation method of a Tonna sinensis extract for reducing a bloodglucose level, comprising steps of: drying a leaf of a Tonna sinensis;pulverizing the leaf as particles; and extracting the particles with asupercritical carbon dioxide to obtain the T. sinensis extract.
 2. Themethod according to claim 1, wherein the leaf is one selected from agroup consisting of a young leaf, a matured leaf, a young shoot and acombination thereof.
 3. The method according to claim 1, wherein theleaf has an aqueous capacity less than 30% after the leaf is dried. 4.The method according to claim 1, wherein the particles have a size lessthan 5 μm in diameter.
 5. The method according to claim 1, wherein theextracting step is performed at a pressure ranged between 350 bar and550 bar and a temperature between 40° C. and 60° C.
 6. A Toona sinensisextract for reducing a blood glucose level comprising at least one of amonoterpene, a monoterpene derivative, a diterpene derivative, atriterpene, a sesquiterpene, sequiterpene derivative, a saturated fattyacid, a unsaturated fatty acid, an alkane, an alkene and a phytosterol.7. The extract according to claim 6, wherein the monoterpene comprisesan α-pinene and an β-pinene, the monoterpene derivative comprises alimonene, the diterpene derivative comprises a phytol, and thetriterpene comprises a squalene.
 8. The extract according to claim 6,wherein the sesquiterpene comprises an α-cubebene, a copaene, anaromadendrene, a caryophyllene, an α-caryophyllene, a β-caryophyllene,an aristolene, a γ-patchoulene, an α-selinene, a β-selinene, aγ-selinene, an elixnen, a germacrene A, a germacrene B, a germacrene D,a β-elemene, a γ-elemene, a δ-elemene, a τ-elemene, a γ-cadiene, aτ-muurolene, an α-farnesene and a lepidozene, and the sesquiterpenederivative comprises a caryophylene oxide, a spathulenol, a ledeneoxide, a germacrene D-4-ol and a globulol.
 9. The extract according toclaim 6, wherein the saturated fatty acid comprises an ethylpentadecanoate and an ethyl palmitate, and the unsaturated fatty acidcomprises a methyl linoleate, an ethyl 9,12-octadecadienoate and anethyl α-linolenate.
 10. The extract according to claim 6, wherein thealkane has a carbon number not less than
 21. 11. The extract accordingto claim 10, wherein the alkane comprises an n-heneicosane, ann-docosane, an n-tricosane, an n-pentacosane, an n-heptacosane, ann-octacosane, an n-nonacosane, an n-hentriacontane, an n-dotriacontane,an n-pentatriacontane, an n-hexatricontane, an n-tritetracontane and ann-tetratetracontane.
 12. The extract according to claim 6, wherein thealkene comprises a (17E)-17-pentatriacontene, and the phytosterolcomprises a β-sitosterol.
 13. The extract according to claim 6, whereinthe extract further comprises a 6-methyl-5-hepten-2-one and a phytone.14. The extract according to claim 6 being prepared by extracting driedparticles of a leaf of a Toona sinensis with a supercritical carbondioxide.
 15. The extract according to claim 14, wherein the leafcomprises one being selected from a group consisting of a young leaf, amatured leaf, a young shoot and a combination thereof.
 16. The extractaccording to claim 6 being manufactured as at least one product of afood and a drug.
 17. The extract according to claim 6 further being usedfor improving metabolic diseases and inhibiting an accumulation of fats.18. A preparation method of a Toona sinensis extract for reducing ablood glucose level, comprising a step of extracting a Toona sinensiswith a supercritical fluid to obtain the T. sinensis extract.
 19. Themethod according to claim 18, wherein the supercritical fluid comprisesa carbon dioxide, the T. sinensis extract comprises a Toona sinensisessential oil, and the T. sinensis has a leaf to be prepared asparticles.
 20. The method according to claim 19, wherein the particlesare obtained by sequentially drying and pulverizing the leaf.
 21. Amethod for detecting at least one component in a Toona sinensis extract,comprising a step of determining an amount of the at least one componentbeing one selected from a group consisting of a monoterpene, amonoterpene derivative, a diterpene derivative, a triterpene, asesquiterpene, sequiterpene derivative, a saturated fatty acid, aunsaturated fatty acid, an alkane, an alkene, a phytosterol,6-methyl-5-hepten-2-one and a phytone using GC-MS.